Clean sugar and lignin from non-chemically pretreated lignocellulosic biomass

ABSTRACT

Methods of producing clean (e.g., low sulfur and metal ion content, and free of fermentation inhibitors) sugar and lignin-rich streams, and downstream conversion products, from lignocellulosic biomass, may include obtaining non-chemically pretreated, milled lignocellulosic biomass, reacting the milled lignocellulosic biomass with an enzymatic agent to produce a slurry that includes converted monomeric lignocellulosic sugars and lignin-rich residuals, and separating the slurry into a sugar stream that includes the converted monomeric lignocellulosic sugars and a lignin-rich stream that includes the lignin-rich residuals. The sugar stream, not including water, includes at least 75% monomeric lignocellulosic sugar, less than 0.20% sulfur, and less than 3.0% metal ion content, and the lignin-rich stream includes at least 35% lignin and less than 0.50% sulfur. Some methods include producing fermentation products such as alcohols and/or organic acids from the sugar stream, and/or use of the lignin residuals in fuels.

TECHNICAL FIELD

This disclosure relates to enzymatic hydrolysis of lignocellulosicbiomass, and in particular to the production of clean (e.g., having lowsulfur and metal ion content, and being free of fermentation inhibitorsand other impurities) sugar and lignin-rich streams, and downstreamconversion products, from non-chemically pretreated, milledlignocellulosic biomass.

BACKGROUND

Lignocellulosic biomass represents an attractive and environmentallyfriendly starting material for production of carbohydrates (such assugars) and downstream processes (such as fermentation to alcohols,organic acids, polymer precursors, etc.), and/or of lignin, since theraw material is obtained from renewable resources. Many non-foodlignocellulosic materials are potential sources, including wood andby-products of wood processing (e.g., chips, sawdust, shavings).

Example methods of producing sugars from lignocellulosic biomass includeacid hydrolysis and enzymatic hydrolysis. In acid hydrolysis, a mixtureof woody material (such as wood chips) in acid is heated in the presenceof water under conditions sufficient to hydrolyze cellulose, producingsugars and lignin residue. US20120264873 of Eyal, et al., discloses anexample of a strong hydrochloric acid hydrolysis method. Acid is notconsumed in the reaction, so the hydrolysis co-products typicallyinclude residual acid content. Additionally, in some acid hydrolysismethods, toxic degradation products are produced, including inhibitorsof downstream fermentation (e.g., furans, such as hydroxymethylfurfural(HMF), furfuraldehyde, etc.). Removal of residual acid and inhibitors,such as by waching, ion exchanging, or other purification methods,represents additional cost and time.

Enzymatic hydrolysis proceeds by breaking cellulose chains into sugarmolecules by suitable enzymes. The cellulose present in lignocellulosicbiomass is recalcitrant to enzyme systems, largely due to the complexstructure of plant cell walls, and generally requires pretreatment tomake the cellulose fraction accessible by enzymatic hydrolysis. Chemicaland organic solvent pretreatments are common, but such techniques aretypically accompanied by significant cost, as well as a host ofenvironmental management and waste treatment issues resulting from, orotherwise related to, use of the pretreatment chemicals. Examplechemical pretreatment methods include sulfur dioxide treatment,oxidative delignification, ozonolysis, ammonia fiber explosion (AFEX),treatments with organosolvents and/or ionic liquids, and so forth (see,e.g., Zheng, et al., Overview of biomass pretreatment for cellulosicethanol production, Int. J. Agric. & Biol. Eng., 2(3), pp 51-68, 2009).Besides material cost and regulatory requirements, like acid hydrolysis,chemical pretreatment methods characteristically introduce residualimpurities into the hydrolysis reaction co-products. Moreover, manychemical pretreatment processes modify the lignin present inlignocellulosic biomass, and/or introduce impurities such as sulfur andsulfur compounds, which collectively create complexity and cost inisolation and/or use of such lignin, particularly in liquid fuelproducts for which low sulfur content is a requirement.

Example non-chemical pretreatment methods for enzymatic hydrolysisinclude steam explosion and liquid hot water pretreatment, both of whichrequire significant energy costs. Moreover, even in these non-chemicalmethods, the use of hot water or steam have been known to result inacetic acid formation, which in turn reacts with hemicellulose sugar toform furfural during pretreatment (Harmsen et al., Literature review ofphysical and chemical pretreatment processes for lignocellulosicbiomass, ECN-E-10-013, Energy Research Centre of the Netherlands, 2010).

Mechanical pretreatment, such as milling wood chips to fine wood powderto the extent that the tightly structured cell wall is opened, canincrease susceptibility to enzymatic hydrolysis by allowing the enzymesto more readily contact the cellulose (see, e.g., Agarwal, et al.,Enzymatic hydrolysis of biomass: Effects of crystallinity, particlesize, and lignin removal, Proceedings of the 16^(th) ISWPFC, China LightIndustry Press, 2011). However, typical milling techniques often requiresignificant energy cost and/or time to yield a suitable particle size.Moreover, milling is often accompanied by one or more chemicalprocesses, such as to remove pitch or lignin, to more efficientlyachieve a desired particle size and/or powder consistency. Inparticular, delignification of milled biomass prior to enzymatichydrolysis has been found to make the cellulose in milled biomass moreaccessible to the enzyme system used, such as by further increasing thesurface area of the milled material by forming pores via lignin removal(Id.; see also WO2010077170 of Davidov et al.).

SUMMARY

The current disclosure describes methods of clean (e.g., free offermentation inhibitors and other chemical residuals), low-sulfurlignocellulosic sugar and lignin-rich residual production from enzymatichydrolysis performed on lignocellulosic biomass without chemicalpretreatment, and products from such methods. Clean lignocellulosichydrolysis products present fewer process and cost obstacles in variousdownstream applications, such as conversion of lignocellulosic sugar toalcohol, organic acids, polymer precursors, and so forth, and/or use oflignin-rich residuals.

An illustrative example method of lignocellulosic biomass conversionincludes obtaining non-chemically pretreated, milled lignocellulosicbiomass having a particle size of about 300 microns or less, reactingthe milled biomass with an enzymatic agent (such as by means ofenzymatic hydrolysis) to produce a slurry that includes convertedmonomeric lignocellulosic sugars and lignin-rich residuals, andseparating the slurry into a sugar composition that includes theconverted monomeric lignocellulosic sugars and a lignin-rich compositionthat includes the lignin-rich residuals. In contrast to methodsincorporating chemical pretreatment, the slurry produced according tothe methods of the present disclosure is free of furans and otherfermentation inhibitors, as are the hydrolysis reaction co-products,i.e., the sugar composition and the lignin-rich composition. The sugarcomposition includes at least 75% monomeric lignocellulosic sugar andless than 0.20% sulfur, and less than 3.0% metal ion content after wateris removed, and the lignin-rich composition includes at least 35% ligninand less than 0.5% sulfur.

In some methods, the biomass is milled, and/or otherwise processed priorto milled, such as by chipping, resizing, drying, etc., for example ifthe biomass is wood (such as softwood). Such processing steps willrelate to the type of biomass used.

In some methods, separating the co-products is accomplished by means offiltering. In some methods, the slurry may additionally includeundigested lignocellulosic biomass and/or unused enzymatic agent. In thelatter case, some methods may include recycling the enzymatic agent,such as by adding fresh, or unreacted, lignocellulosic biomass to theslurry, for example to effect an additional hydrolysis reaction.

Some methods may further include drying or otherwise concentrating oneor both of the sugar composition and the lignin-rich composition, suchas by dewatering the sugar composition to produce a cleanlignocellulosic sugar syrup, powder, or crystalline mixture, and/ordrying the lignin-rich composition to achieve a solids content of adesired amount, such as 80% or above.

Some methods may further include downstream processing of the sugarcomposition and/or the lignin-rich composition, or concentrated versionsthereof, such as by converting the lignocellulosic sugar composition toalcohol (or organic acid, or polymer precursor, etc.), suspending thelignin-rich composition in a fuel, and so forth.

Another illustrative example method of hydrolytic lignocellulosicbiomass conversion in accordance with the present disclosure includes,in a first hydrolysis reaction, reacting milled lignocellulosic biomasswith an enzymatic agent to produce a first slurry that includesconverted lignocellulosic sugars, lignin-rich residuals, and unusedenzymatic agent, then mixing additional milled lignocellulosic biomasswith the first slurry to produce a slurry mixture, such as in acontinuous mixing tube or tubular reactor outside the first hydrolysisreaction. The lignocellulosic sugars are then separated from the slurrymixture. In a second hydrolysis reaction, the milled lignocellulosicbiomass in the slurry mixture is reacted with the enzymatic agent in theslurry mixture to produce a second slurry that includes convertedlignocellulosic sugars and lignin-rich residuals. The convertedlignocellulosic sugars are then separated from the second slurry. Theseparated sugars may then be combined, further filtered, andconcentrated for downstream fermentation and/or other uses as indicatedabove.

Illustrative embodiments of a lignin-rich composition produced fromlignocellulosic biomass according to the present disclosure include atleast 40% lignin by weight and has an HHV of at least 9000 BTU/lb.Illustrative embodiments of sugar compositions produced fromlignocellulosic biomass according to the present disclosure include atleast 75% monomeric lignocellulosic sugar and have less than 0.20%sulfur content.

The concepts, features, methods, and component configurations brieflydescribed above are clarified with reference to the accompanyingdrawings and detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart schematically representing an illustrativeexample of a biomass conversion process in accordance with the presentdisclosure.

FIG. 2 is a flow chart schematically representing a further illustrativeexample of a biomass conversion process in accordance with the presentdisclosure.

FIG. 3 is a graph showing monomeric sugar profiles of an example sugarstream prepared by performing enzymatic hydrolysis on a milled Douglasfir wood sample in accordance with the present disclosure.

FIG. 4 is a graph showing the total ethanol titer and the total sugarconsumption during ethanol fermentation of the example sugar streamshown in FIG. 3.

FIG. 5 is a graph showing lactic acid accumulation in a lactic acidfermentation process performed on the example sugar stream shown in FIG.3.

DETAILED DESCRIPTION

Lignocellulosic biomass represents an attractive and environmentallyfriendly starting material for production of carbohydrates (such assugars) and downstream processes (such as fermentation to alcohols)since it is obtained from renewable resources. Many non-foodlignocellulosic materials are potential sources, including wood (bothhardwood and softwood), by-products of wood processing (e.g., chips,flakes, sawdust, shavings, and other wood residuals), as well as otheragricultural residues, herbaceous energy crops, and so forth. In theillustrative embodiments and examples discussed herein, wood, and inparticular softwood, is used as lignocellulosic biomass, in part due tolow cost and high availability.

Compositions of various types of biomass vary, but generally includecellulose as the major component, followed by hemicellulose and lignin,and various proteins, oils, and ash.

Pretreatment, as the term is used in the context of enzymatichydrolysis, refers to a process to prepare biomass to facilitatebioconversion, such as a process that converts biomass from its nativeform into a form suitable for cellulose hydrolysis. Several chemicalpretreatments summarized above, while effective, result in chemicalresidue and/or lignin impurification in hydrolysis products, as well asrequire often significant material cost, and also represent health andenvironmental concerns.

Mechanical pretreatment of biomass, such as milling, especially to aparticle size on the order of hundreds or even tens of microns, providesa material characterized by a vastly increased surface area and porosity(as compared with untreated biomass), and higher availability of thecellulose to enzymes. As noted above, some milling techniques commonlyinclude solvent treatment steps, such as solvent extraction, to removepitch, lignin, and/or other organic compounds prior to enzymatichydrolysis. WO2010077170 of Davidov et al. provides such an example.However, the methods of the present disclosure employ no chemicalpretreatment.

Referring to the drawings, two illustrative and non-exclusive examplesof biomass conversion processes are somewhat schematically indicated inFIGS. 1 and 2 to include a series of interconnected boxes that representprocess steps. As will be evident from the description below, one ormore of the represented process steps may further include multiplesub-steps or processes. As such, the represented steps are notexhaustive or exclusive of other steps and methods that may beconsistent with those indicated in FIG. 1. Further, some methods mayomit one or more of the represented steps, include steps notrepresented, combine steps, perform steps in a different order than asindicated, etc. The steps may be carried out in any suitable system orsystems by one or more components, which may be co-located in the samefacility or located in different facilities, and so forth.

In FIG. 1, the example biomass conversion process is shown to includepreparation steps including wood chipping at 100, resizing at 110,drying at 120, and milling at 130. Such steps are optional to themethods disclosed herein, may vary depending on the nature of thebiomass used, and may be performed according to suitable techniques. Forexample, milling is typically performed in one or more ball mills, suchas in succession (e.g., coarse to fine), a tandem mill, and so forth. Inthe methods of the present disclosure, fine wood powder having aparticle size of less than 300 microns (and in some cases less than 25microns, or less than 10 microns) is obtained by use of a tandem milland without chemical pretreatment. Optionally, the milling step mayinclude screening, such as to isolate a desired particle size range.

Once non-chemically pretreated, milled lignocellulosic biomass having apreferred particle size is obtained, an enzymatic agent is allowed toreact with the biomass under conditions suitable to effect cellulosehydrolysis, at 140. For example, the biomass sample is typicallysuspended in water, to which an aqueous solution of enzymatic agent isadded, under suitable temperature and pH. Because fine biomass watersrapidly and tends to go down immediately (as compared with conventionalwood chips, which float), water needs and hydrolysis preparation timecan be minimized. The enzymatic agent, usually one or more enzymes orenzyme complexes of enzymes, such as cellulase enzymes and other enzymesuseful in hydrolysis of polysaccharides (polymer sugars), is typicallyselected to match or otherwise target some or all of the polysaccharidecomponents of the raw material. For example, in the examples discussedbelow, Douglas fir wood powder is used, and an enzymatic agent thatincludes enzyme complexes known to be suitable for conversion oflignocellulosic polysaccharides is used, e.g., CTec2 and HTec2 enzymeproducts available from Novozymes A/S, in appropriate ratios and undersuitable temperature, pH, and other reaction conditions. Accordingly,the hydrolysis step may include temperature and/or pH control. Forexample, in the lab-scale experiments described below, a buffer (e.g.,sodium citrate) is added to the hydrolysis solution and the pH isperiodically adjusting (e.g., with NaOH). On an industrial scale, pH maybe controlled by automated base or acid addition lines.

Hydrolysis produces a slurry consisting of a hydrolyzed product thatincludes converted monomeric lignocellulosic sugars, as well aslignin-rich residuals, and which may also include undigested rawmaterials (e.g., milled biomass) and/or unused enzymatic agent. Theslurry is clean, that is, the slurry is free of furans and otherfermentation inhibitors, and extremely low in sulfur content, as aresult of the non-chemical pretreatment of the raw materials. At 150 and160, the slurry is separated into a clean sugar composition (whichincludes the converted monomeric lignocellulosic sugars) and a cleanlignin-rich composition (which includes the lignin-rich residuals andother components, if any). In the example process, the separation isaccomplished by means of filtration, and in particular a coarsefiltration at 150, followed by a fine filtration at 160, in which thesugar composition is the filtrate. The filtrate contains water,converted monomeric lignocellulosic sugars, and other componentsincluding some amount of water soluble wood extractives, and a smallamount of insolubles, such as undigested hemicellulose, cellulose,lignin and wood extractives, which in total are about 3-7% (or less) ofthe total monomeric sugar content. However, in methods consistent withthe present disclosure, separation may be achieved by any suitablemethod, which may or may not include one or more filtration steps.

The sugar composition, or clean sugar stream, generated at 170 from theslurry, is aqueous, and is typically characterized by a monomericlignocellulosic sugar titer of about 8-10% (wt/vol), a sulfur contentless than about 0.02% (wt/wt), a metal content less than about 0.5%(wt/wt), and no furan content. Not including the water content, thesugar composition of the clean sugar stream ranges from about 85-95%(wt/wt), with the sulfur content less than 0.20% (wt/wt), and withsoluble wood extractives and insoluble organic compounds such asundigested cellulose, hemicelluloses, lignin, wood extractives, and soforth, accounting for about 3-7% (wt/wt).

Optionally, a concentrating step (at 180), and/or a drying orcrystallization step (at 190) may be employed to produce a clean sugarsyrup (at 200) or a clean dry sugar mixture (at 210), such as by anysuitable technique(s).

The solid stream produced, at 220, from the filtration (at 150 and 160)includes the lignin-rich composition, in which the sulfur content isless than 0.1% by weight. Although not shown, the lignin-richcomposition may be subjected to further processing, such as washing (toremove unreacted biomass and/or enzymes), dewatering (to increase solidscontent), and so forth.

A second illustrative and non-exclusive example of a biomass conversionprocess is somewhat schematically indicated in FIG. 2. The process inFIG. 2 includes many of the same process steps as described above, butincorporates modifications that integrate biomass residual and enzymerecycling into the process. To the extent that various steps representedin FIG. 2 are similar to those described above with respect to FIG. 1,they are summarized in the paragraphs below.

Milled lignocellulosic biomass such as wood powder may be prepared in aseries of preparation steps, for example wood chipping (at 300), chipresizing (at 310), chip drying (at 320) and milling (at 330), in whichthe wood is milled into wood powder. As noted above, the preparation ofthe biomass is shown not to include any chemical pretreatment. Onceobtained, the milled biomass is hydrolyzed in a first hydrolysisreaction, at 340, by hydrolytic enzymes to produce a slurry thatincludes monomeric lignocellulosic sugars (i.e., glucose, xylose,mannose, arabinose and galactose), while no fermentation inhibitors areformed, e.g. furans such as hydroxymethylfurfural and furfuraldehyde.

The slurry is transported from the first hydrolysis reaction 340, whichmay take place in a suitable reaction tank or vessel (“hydrolysisunit”), to a filtration unit or suitable separation process, such as bymeans of a mixing tube 345 or appropriate conduit. A new milled biomassstream is continuously added to the mixing tube 345 to mix with theslurry transported from the first hydrolysis reaction, such as by meansof a static mixer or otherwise. As such, the mixing tube allows mixingof the enzyme in the slurry and the binding of the unused enzymes to thenew milled wood. This “slurry mixture,” which includes the undigestedwood residuals and the new milled wood bound with enzyme, is transportedto a separation process, shown as two separate filtration units 350 and360, although a different separation process configuration may be used.In filtration unit 350, a course filtration process separates the solidof the undigested wood residuals and the new milled wood bound withenzymes away from the liquid stream containing monomeric sugars. In asecond hydrolysis reaction conducted at 420, less enzymatic agent isneeded per unit of undigested raw material due to the enzyme recyclingstep in 345 and 350. The second hydrolysis reaction 420 produces asecond slurry, which is sent to another course filtration unit 430,which separates out the low sulfur lignin-rich residuals, at 440. Thefiltrate from course filtration unit 430 is transported to finefiltration unit 360 for a fine filtration, to produce a low sulfur andno-furan clean lignocellulosic sugar stream, indicated at 370. After370, a concentrating step (at 380) may produce a low sulfur and no-furanclean lignocellulosic sugar syrup, at 400. Further, an optional dryingstep (at 390) may produce a low sulfur and no-furan lignocellulosic drysugar product, at 410.

Although not shown in the drawings, methods according to the presentdisclosure may optionally include any of several downstream processesperformed on the clean sugar and/or lignin products prepared as above.For example, the clean sugar stream and/or concentrated products thereof(syrups, dry mixtures, and so forth) may be converted to alcohols orother products by suitable fermentation techniques. Accordingly, methodsmay include producing alcohol from the converted monomericlignocellulosic sugars in the sugar composition. However, because thereis no furan inhibitor present in the clean sugar stream, there is noneed to remove inhibitor prior to fermentation, such as would be thecase with standard chemical pretreatment methods including acidhydrolysis. Fermentation methods in accordance with the presentdisclosure may include, as a preliminary step, clarification of thesugar composition (or a concentrated version thereof), such as byfiltering, centrifuging, etc., followed by inoculation with anappropriate fermentation agent, such as a yeast strain chosen to use oneor more of the sugars present in the sugar composition for fermentation.For example, C6 sugars such as glucose and mannose may readily beconverted to ethanol using various baker's yeast strains. Accordingly,target fermentation alcohol products may also include isobutanol, and soforth. Reaction conditions such as temperature, pH, time, and so forth,would be as suitable for the target fermentation alcohol product(s)according to standard methods.

Another example of a product that may be produced from the clean sugarstream and/or concentrated products is lactic acid. Accordingly, methodsthat include producing lactic acid from the converted monomericlignocellulosic sugars in the sugar composition may includeclarification of the sugar composition followed by addition of anappropriate fermentation agent and, if appropriate, other reagentssuitable to create a fermenting medium. An example fermentation agentfor lactic acid formation is Lactobacillus rhamnosus, which may beinoculated in a fermentation medium in the presence of the clean sugarstream under suitable conditions to effect lactic acid fermentation.

Conversion of sugars to other downstream products, including otherorganic acids, polymer precursors, and so forth, are known in the art,and methods according to the present disclosure may include additionalsteps directed to producing such products from the sugar compositionsproduced herein. The absence of inhibitors and impurities in the sugarcompositions provides several process advantages in such conversionprocesses, such as by not requiring interim purification steps.

As another example, the lignin-rich residuals may be dewatered and/orsubjected to additional steps to purify the lignin. After the enzymatichydrolysis and the filtration process as described in FIGS. 1 and 2, alignin-rich residual stream is produced. This lignin-rich streamnormally has a moisture content of about 70-90% (or a solid contentranging from 10-30%). This high moisture stream may be dewatered, suchas by pressing or screw-pressing, to a much higher solid content, suchas 40-60%. After the pressing step, the lignin-rich stream is furtherdried in dryer, e.g. a rotary drum dryer, or other drying devices. Oncethe lignin is dried to about or higher than 80% solids, this lignin-richstream can be pelletized in a pelletizer to make a solid fuel. Thissolid fuel can be used and burnt for energy generation and/or steamgeneration in a co-firing boiler with biomass, a biomass boiler, adrying kiln, and a household wood stove, so forth.

The physical form of the dried lignin may be as suitable for theapplication. For example, the dried lignin-rich residuals may milled tovery fine particles, such as 5-25 microns. The milled and finelignin-rich powder can then be used and sprayed as a powder fuel as acombustion fuel in a co-firing boiler with biomass, a biomass boiler, adrying kiln, and so forth. Since the lignin-rich particles are veryfine, the micron size lignin-rich particles can be used as an additivein a liquid fuel, such as a biodiesel, a diesel, and an alcohol.

The following examples summarize representative experiments ofconverting non-chemically pretreated milled lignocellulosic biomass inaccordance with the methods and concepts discussed above.

Example 1. Douglas Fir Wood Chip Preparation and Composition

The softwood wood chips used in this example are Douglas fir wood chips(debarked) from the Weyerhaeuser Longview Pulp Mill in Washington, U.S.The wood chips were first dried to contain less than 10% moisture. Thecarbohydrate composition of Douglas fir chips was determined byconverting the polymeric sugars in the wood chips into monomeric sugarssuch as glucose, xylose, mannose, arabinose and galactose. Table 1 showsthe original polymeric sugar composition of the Douglas fir wood chips.The total polymeric sugar composition for the debarked Douglas fir chipsample is 59.07% (wt/wt). The lignin content of the Douglas fir sampleis 27.10% (wt/wt).

TABLE 1 Douglas fir chip carbohydrate composition Polymer sugarComposition (% wt/wt) Arabinan 1.01 Galactan 2.53 Glucan 38.87 Xylan3.90 Mannan 11.77 Total 59.07

Example 2. Douglas-Fir Wood Chip Milling and Milled Wood PowderComposition

The Douglas fir chips were milled down to wood powder in a tandem millby the Kenko Corporation, Tokyo. The carbohydrate composition of themilled Douglas fir wood powder is shown in Table 2. The total polymericsugar composition for the milled Douglas fir wood powder is 61.00%(wt/wt).

TABLE 2 Milled Douglas fir powder carbohydrate composition Polymer sugarComposition (% wt/wt) Arabinan 1.00 Galactan 2.66 Glucan 41.20 Xylan3.74 Mannan 12.40 Total 61.00

The milled Douglas fir wood powder was diluted in deionized water withsonication to break apart the aggregated particles. Two samples wereplated on glass slides. After drying, each sample slide was imaginedunder scanning electron microscope. The particle sizes of two sampleswere analyzed, and the distributions were found to include about 95-99%of the particles having a size (diameter) smaller than about 25 microns.About 87-93% of the particles of the two samples had a size (diameter)less than about 10 microns. All of the particles in the two samples wereunder 300 microns in size.

Example 3. Clean Sugar Stream Production from Douglas Fir Wood PowderHydrolysis

The milled Douglas fir wood powder from Example 2 was hydrolyzed at 20%consistency with 4.7% (wt/wt) of CTec2 enzyme product (a cellulaseenzyme) along with 0.4% (wt/wt) of HTec2 enzyme product (a xylanaseenzyme), both from Novozymes A/S of Bagsvaerd, Denmark. The hydrolysistests were conducted in 150 ml volume in 250 ml Erlenmeyer shake flasks.The hydrolysis pH (4.8-5.3) of the milled wood slurry was buffered by a100 mmol sodium citrate buffer with periodic pH adjustment with a NaOHsolution. The hydrolysis temperature was controlled at 50° C. andagitation was done by shaking the flask at 200 rpm in an orbitalenvironmental shaking incubator.

After the slurry hydrolysis, the watery slurry mainly consists ofmonomeric lignocellulosic sugars and undigested residuals containinghemicellulose, cellulose and wood lignin. The monomeric sugar profilesof the milled Douglas fir wood (at a consistency of 20%) hydrolysis isshown in FIG. 3. The hydrolysis of the milled Douglas fir wood leveledoff after three days of hydrolysis to achieve a total sugar titer of8.2%. The total sugar titer at the end of the hydrolysis was 8.9%(wt/vol) after about 9 days of hydrolysis. Table 3 shows the monomericsugar titers of each sugar in the end of the hydrolysis.

TABLE 3 Monomeric sugar titers in hydrolyzed Douglas fir wood powder(20% consistency) Monomeric sugar Titer (% wt/vol) Arabinose 0.03Galactose 0.09 Glucose 7.01 Xylose 0.36 Mannose 1.41 Total 8.90

No fermentation inhibitors such as hydroxymethylfurfural and furfuralwere found in the hydrolysis, as shown in Table 4.

TABLE 4 Furan titers in hydrolyzed Douglas fir wood powder (20%consistency) Furan Titer (% wt/vol) Hydroxymethylfurfural 0.000 Furfural0.000 Total 0.000

The sulfur content of the hydrolyzed Douglas fir wood powder slurry wasanalyzed, and the result is shown in Table 5. The wood milling processis a non-chemical and non-sulfite pretreatment method, and therefore,the sulfur content is extremely low. Without being bound by theory, thesulfur content is thought be carried from the original wood composition.The sulfur titer in the filtrate of the hydrolyzed milled wood is 0.016%(wt/wt). Samples 1 and 2 are from different milled Douglas fir samples.

TABLE 5 Sulfur content of filtrate of hydrolyzed milled Douglas fir woodpowder Sample Sulfur Titer (% wt/wt) 1 0.016 2 0.016

The hydrolysis residuals from the above hydrolysis of the milled Douglasfir wood powder were analyzed for the lignin content, higher heatingvalue and sulfur content. Douglas fir wood has a lignin content of about27.10% (wt/wt). Table 6 shows that the hydrolysis residuals of milledDouglas fir wood have higher lignin content compared to the originalDouglas fir wood chips, as well as high BTU HHV values and low sulfur.

TABLE 6 Characteristics of residuals from milled Douglas fir wood powderhydrolysis Lignin HHV Sulfur Sample (% wt/wt) (BTU/lb residuals) (%wt/wt) 1 45.6 9060 0.068 2 54.3 9596 0.055

The clean sugar stream was analyzed for metal and inorganic elements,and the results are shown in Table 7 below. The major metal and someinorganic element content in the clean sugar stream was 0.50% (wt/wt)and 0.39% (wt/wt), respectively, for clean sugar stream sample 1 andsample 2. The sodium citrate buffer contributed significant sodiumcontent to the samples (a debarked softwood chip normally only hassodium content around 50 mg/kg wood). Besides the sodium from the sodiumcitrate buffer, sodium hydroxide used for pH adjustment contributed tosodium level of 0.1% to 0.2% of the total element level in the cleansugar stream.

TABLE 7 Element titer in clean sugar stream samples from hydrolyzedDouglas fir wood powder 1 2 Sample (mg/kg clean (mg/kg clean Elementsugar stream) sugar stream) Ag — — Al 6.3 3.4 As — — B 0.5 0.4 Ba 1.31.0 Be — — Bi — — Ca 142.1 91.3 Cd — — Co — — Cr 0.2 0.2 Cu 2.2 1.1 Fe149.4 156.6 K 310.3 224.5 Li — — Mg 13.5 9.9 Mn 5.2 4.3 Mo 0.0 0.0 Na4,307.9 3,380.3 Ni 0.1 0.1 P 17.3 13.5 Pb 0.0 — Sb — — Se — — Sn — — Sr0.5 0.4 TI — — V — — Zn 5.8 2.9 Total Element 4,962.7 3,890.0 Content0.50% (wt/wt) 0.39% (wt/wt) Note: ″-″ indicates content (if any) isbelow detection limit at analysis.

When water is evaporated from the clean sugar stream, concentrated cleansugar streams are produced. Titers of lignocellulosic sugar monomers,metals and inorganic elements, and sulfur content of exampleconcentrates are shown in Table 8. The total titer is less than 100%,which is balanced with water and other components (including some amountof water soluble wood extractives, and some small amount of insolubles,such as undigested hemicellulose, cellulose, lignin and woodextractives, which in total are about or less than 3-7% of the totalmonomeric sugar content). The sugar composition (water not included) inthe clean stream ranges from 86.8 to 90.9%. Fine filtration may be usedto remove such components. Although not tested, it can be calculatedthat if about 50% of such components are removed by a fine filtration,the sugar composition (water not included) in the clean stream willincrease to 89.5 to 92.2%.

TABLE 8 Sugar titer and composition in clean sugar stream andconcentrates Concentrating factor  2.81  5.62  9.66 Items Original TiterProduct 1 Product 2 Product 3 Sugar titer (%) 8.90  25.00 50.00 86.00Major metal and 0.16-0.27 0.45-0.76 0.90-1.52 1.55-2.61 inorganic (%)Sulfur (%) 0.016   0.045   0.090   0.153 Other components 0.27-0.620.75-1.75 1.50-3.50 2.58-6.02 (%) Sugar Composition 86.8-90.9 (%), waternot included

Example 4. Clean Sugar Stream Fermentation to Ethanol

A clean sugar stream from the above hydrolyzed wood slurry in Example 3was prepared by a clarification in a centrifuge to remove the coarseresiduals and a filtration through a 0.45 micron filter to remove thefiner particles large than 0.45 micron. The 0.45 micron filtration alsoserved as a means to sterilize the sugar stream for the subsequentfermentation tests. The clean sugar stream is transparent and has alight amber color, showing no apparent particulates. The clean sugarstream was concentrated in a vacuum evaporator to form a high sugartiter syrup in order to achieve a high ethanol titer.

The ethanol fermentation medium was made from the clean sugar streamsyrup supplemented with 0.5% corn steep liquor and 0.07% urea. Theinitial total sugar titer was 23.1% (wt/vol). An overnight grown baker'syeast strain NABC XR was inoculated at 2 g/L in the ethanol fermentationmedium in a 50 ml shake flask. This yeast strain uses C6 sugar forethanol fermentation, i.e., glucose and mannose were used, but notxylose and arabinose. The fermentation was conducted in an orbitalshaking incubator at 33° C. and 100 rpm. The initial fermentation pH was5.0. During fermentation, the pH was checked twice daily and readjustedback to pH 4.5-5.0 with a 20% KOH solution. FIG. 4 shows the totalethanol titer and the total sugar consumption during the ethanolfermentation. At the end of the 3-day fermentation, an ethanol titer of9.8% (wt/vol) was achieved.

Example 5. Clean Sugar Stream Fermentation to Lactic Acid

The clean sugar stream from Example 4, without further sugarconcentrating, was used as the carbon source for lactic acidfermentation. A lactic acid fermenting bacteria strain, Lactobacillusrhamnosus ATCC11443, was obtained from American Type Culture Collection(ATCC). The fermentation medium contains the clean sugar streamsupplemented with nutrients 0.87 g/L peptone, 0.43 g/L yeast extract,0.004 g/L MnSO₄.H₂O, 0.17 g/L NaH₂PO₄, 0.36 g/L MgCl₂.6H₂O and 0.17 g/L(NH₄)₂SO₄. The fermentation medium had an initial pH of 6.5. The mediumwas filter-sterilized through a 0.45 micron filter cup. Culture tubes(20 ml size) and rubber septa were sterilized at 250° F. for 15 minutesbefore lactic acid fermentation. A seed culture medium with all thenutrients and 4% glucose was used to propagate the Lactobacillusrhamnosus strain. The lactic acid fermentation was conducted in a totalvolume of 11.5 ml in 20 ml anaerobic culture tubes. After the medium wasdispensed to each tube, the tube was sealed with rubber septa with acrimper. The headspace of each tube was purged with 5% hydrogen and 5%carbon dioxide balanced with nitrogen.

The Lactobacillus rhamnosus seed culture was grown for 24 hours in thesame nutrient medium with 4% glucose. The ready Lactobacillus seed wascentrifuged down and re-suspended in 1 ml nutrient medium without sugarand used as the seed culture. The fermentation medium was inoculatedwith 0.5 ml seed culture. The fermentation was conducted in an incubatorat 37° C. The fermentation pH was checked daily and readjusted to pH5.0-6.0 with a NaOH solution.

The Lactobacillus rhamnosus was very active in lactic acid fermentation.The fermentation dropped quickly to less than pH 4.5 daily and wasreadjusted back to pH 5.0 to 6.0 daily. FIG. 5 shows the lactic acidaccumulation in the fermentation. At the end of day 4, the lactic acidachieved 5.2% (wt/vol). This test showed that the clean sugar streamfrom milled Douglas fir wood supported well the bacterial lactic acidfermentation by Lactobacillus rhamnosus.

Example 6. Suspension of Lignin-Rich Residuals Particles in a LiquidFuel

To test suitability for incorporation of the lignin-rich residuals in aliquid fuel, the lignin-rich residuals from Example 3 was dried andmilled with a mortar and pestle to form a fine powder, having a particlesize of about 5-25 microns. Various amount of the lignin residual powderwere mixed with canola oil. The lignin residual particles werewell-suspended in the oil without a quick settling down. The varioussuspensions of lignin in the oil and the calculated HHV of the oil andlignin mixture are shown in Table 9.

TABLE 9 Calculated HHV for suspensions of lignin-rich residuals in oilLignin residual in oil (% wt/wt) Calculated HHV (BTU/lb) 53.3 12,55033.3 13,857 20.0 14,729 14.3 15,102 10.5 15,348 6.8 15,589 0.0 16,000

Depending on BTU value needs, the lignin-rich residuals may be blendedwith a liquid fuel and the resulting lignin-rich residual and oilmixture still have high HHV value, and may be suitable for use as liquidfuel.

Illustrative, non-exclusive examples of descriptions of some methods andcompositions in accordance with the scope of the present disclosure arepresented in the following numbered paragraphs. The following paragraphsare not intended to be an exhaustive set of descriptions, and are notintended to define minimum or maximum scopes, or required elements orsteps, of the present disclosure. Rather, they are provided asillustrative examples of selected methods and compositions that arewithin the scope of the present disclosure, with other descriptions ofbroader or narrower scopes, or combinations thereof, not specificallylisted herein still being within the scope of the present disclosure.

A. A method of lignocellulosic biomass conversion, the methodcomprising:

obtaining non-chemically pretreated, milled lignocellulosic biomasshaving a particle size less than about 300 microns;

reacting the milled lignocellulosic biomass with an enzymatic agent toproduce a slurry that includes converted monomeric lignocellulosicsugars and lignin-rich residuals; and

separating the slurry into a sugar composition that includes theconverted monomeric lignocellulosic sugars and a lignin-rich compositionthat includes the lignin-rich residuals;

wherein the sugar composition, not including water, includes at least75% monomeric lignocellulosic sugar, less than 0.20% sulfur, and lessthan 3.0% metal ion content; and

wherein the lignin-rich composition includes at least 35% lignin andless than 0.50% sulfur.

A.1. The method of paragraph A, wherein obtaining milled lignocellulosicbiomass includes milling lignocellulosic biomass.

A.1.1. The method of paragraph A or A.1, further including one or moreof chipping, resizing, and drying lignocellulosic biomass, prior tomilling.

A.2 The method of any of paragraphs A through A.1.1, wherein thelignocellulosic biomass is wood.

A.2.1. The method of any of paragraphs A through A.2, wherein the woodis softwood.

A.3 The method of any of paragraphs A through A.2.1, wherein the milledlignocellulosic biomass has a particle size less than about 25 microns.

A.3.1 The method of any of paragraphs A through A.3, wherein the milledlignocellulosic biomass has a particle size less than about 10 microns.

A.4 The method of any of paragraphs A through A.3.1, wherein the slurryis free of furans and other fermentation inhibitors.

A.5 The method of any of paragraphs A through A.4, wherein separatingthe slurry includes filtering the slurry to yield a filtrate thatincludes the sugar composition, and a high solid stream that includesthe lignin-rich composition and undigested lignocellulosic biomass.

A.6 The method of any of paragraphs A through A.5, further comprising,subsequent to separating the slurry, dewatering the sugar composition toproduce a concentrated lignocellulosic sugar mixture.

A.6.1 The concentrated lignocellulosic sugar mixture produced accordingto the method of paragraph A.6.

A.6.2. A dry lignocellulosic sugar mixture produced according to themethod of paragraph A.6.

A.7 The method of any of paragraphs A through A.6.1, further comprising,subsequent to separating the slurry, drying the lignin-rich compositionto produce a lignin composition having a solid content of at least 80%.

A.7.1 The lignin composition produced according to the method ofparagraph A.7.

A.8 The method of any of paragraphs A through A.7.1, wherein reactingthe milled lignocellulosic biomass with an enzymatic agent includesrecycling the enzymatic agent.

A.8.1. The method of paragraph A.8,

wherein the slurry includes unused enzymatic agent; and

wherein recycling the enzymatic agent includes adding fresh, unreactedmilled lignocellulosic biomass to the slurry.

A.8.1.1. The method of paragraph A.8.1, wherein adding milledlignocellulosic biomass to the slurry is performed prior to separatingthe slurry.

A.8.1.2. The method of paragraph A.8.1,

wherein separating the hydrolysis product mixture includes filtering theconverted monomeric lignocellulosic sugars from the slurry to leave thelignin-rich residuals, undigested lignicellulosic biomass, and unusedenzymatic agent.

A.9 The method of any of paragraphs A through A.8.1.2, wherein thecomposition of the sugar composition, not including water, includes atleast 85.0% (w/w) monomeric lignocellulosic sugar.

A.9.1 The method of any of paragraphs A through A.9, wherein thecomposition of the sugar composition, not including water, includes atleast 90.0% (w/w) monomeric lignocellulosic sugar.

A.10. The method of any of paragraphs A through A.9.1, wherein thecomposition of the sugar composition, including water, has a sugar titerof at least 8.9% (wt/vol).

A.11 The method of any of paragraphs A through A.10, wherein thecomposition of the sugar composition, including water, has a metal ioncontent of less than about 0.50% (wt/wt).

A.12 The method of any of paragraphs A through A.11, wherein thecomposition of the sugar composition, including water, has a sulfurcontent of less than about 0.02% (wt/wt).

A.13 The sugar composition produced by the method of any of paragraphs Athrough A.12.

A.14 The lignin-rich composition produced by the method of any ofparagraphs A through A.12.

A.15 The method of any of paragraphs A through A.14, further comprisingproducing an alcohol fermentation product from the converted monomericlignocellulosic sugars in the sugar composition.

A.15.1 The method of paragraph A.15, wherein the alcohol fermentationproduct includes ethanol.

A.15.2. The method of paragraph A.15, wherein the alcohol fermentationproduct includes isobutanol.

A.15.2. The alcohol produced by the method of any of paragraphs A.15through A.15.2.

A.16 The method of any of paragraphs A through A.15, further comprisingproducing an organic acid fermentation product from the convertedmonomeric lignocellulosic sugars in the sugar composition.

A.16.1 The method of paragraph A.16, wherein the organic acidfermentation product includes lactic acid.

A.16 The method of any of paragraphs A through A.14, further comprisingproducing lactic acid from the converted monomeric lignocellulosicsugars in the sugar composition.

B.1 A method of hydrolytic lignocellulosic biomass conversion, themethod comprising:

in a first hydrolysis reaction, reacting non-chemically pretreated,milled lignocellulosic biomass to with an enzymatic agent to produce afirst slurry that includes converted lignocellulosic sugars, lignin-richresiduals, and unused enzymatic agent;

mixing additional non-chemically pretreated, milled lignocellulosicbiomass with the first slurry to produce a slurry mixture;

separating the converted lignocellulosic sugars from the slurry mixture;

in a second hydrolysis reaction, reacting the milled lignocellulosicbiomass in the slurry mixture with the unused enzymatic agent in theslurry mixture to produce a second slurry that includes convertedlignocellulosic sugars and lignin-rich residuals; and

separating the converted lignocellulosic sugars from the second slurry.

B.1 The method of paragraph B,

wherein the first hydrolysis reaction is carried out in a hydrolysisunit;

wherein the separating is carried out in a filtration unit; and

wherein the mixing is carried out while transporting the slurry mixturefrom the hydrolysis unit to the filtration unit.

B.1.1 The method of paragraph B or B.1, wherein the mixing is performedcontinuously.

B.2. The method of any of paragraphs B through B.1.1, further includingcombining the converted lignocellulosic sugars separated from the firstand second slurries.

B.3 The method of any of paragraphs B through B.2, further includingadding additional enzymatic agent to the second hydrolysis reaction.

B.4 The method of any of paragraphs B through B.3, further includingcombining the lignin-rich residuals from the first and second slurries.

C.1 A lignin-rich composition produced from lignocellulosic biomass, thelignin-rich composition comprising at least 40% lignin by weight andhaving an HHV of at least 9000 BTU/lb.

C.1 The lignin-rich composition of paragraph C, wherein the sulfurcontent is less than 0.1% by weight.

C.2 The lignin-rich composition of paragraph C or C.1, comprising atleast 45% lignin by weight.

C.3 The lignin-rich composition of any of paragraphs C through C.2,comprising at least 50% lignin by weight.

C.4 The lignin-rich composition of any of paragraphs C through C.3,comprising at least 54% lignin by weight.

C.5 The lignin-rich composition of any of paragraphs C through C.4,having an HHV of at least 9500 BTU/lb.

C.6 A liquid fuel mixture including the lignin-rich composition of anyof paragraphs C through C.5.

C.6.1 The liquid fuel mixture of paragraph C.6, wherein the liquid fuelis one or more of a diesel fuel, a biodiesel fuel, and an ethanol fuel.

C.6.2 The liquid fuel mixture of paragraph C.6 or C.6.1, containing5-80% of the lignin-rich composition.

C.6.3 The liquid fuel mixture of any of paragraphs C.6 through C.6.2,wherein the lignin-rich composition has a particle sizes of 5-30microns.

C.6.4 The liquid fuel mixture of any of paragraphs C.6 through C.6.2,wherein the lignin-rich composition has a particle sizes of 10-15microns.

C.7 The use of the lignin-rich composition of any of paragraphs Cthrough C.5 as a solid fuel.

C.8 Any of the lignin-rich compositions of any of paragraphs C throughC.5 produced by the method of any of paragraphs A through B.4.

D. A lignocellulosic sugar composition produced from lignocellulosicbiomass, the sugar composition, not including water, comprising at least75% monomeric lignocellulosic sugar and having a sulfur content lessthan about 0.20%.

D.1. The lignocellulosic sugar composition of paragraph D, wherein thesugar composition, not including water, comprises at least 85% monomericlignocellulosic sugar.

D.2 The lignocellulosic sugar composition of paragraph D or D.1, whereinthe sugar composition, not including water, comprises at least 90%monomeric lignocellulosic sugar.

D.3 The lignocellulosic sugar composition of any of paragraphs D throughD.2, wherein the sugar composition, not including water, includes lessthan 3.0% metal ion content.

D.4 The lignocellulosic sugar composition of any of paragraphs D throughD.3, wherein the sugar composition is aqueous, and has a sugar titer ofat least 8.9% (wt/vol).

D.4.1 The lignocellulosic sugar composition of paragraph D.4, whereinthe aqueous sugar composition has a sulfur content of less than about0.02% (wt/wt).

D.4.2 The lignocellulosic sugar composition of paragraph D.4 or D.4.1,wherein the aqueous sugar composition has a metal ion content of lessthan about 0.50% (wt/wt).

D.5 Any of the lignocellulosic sugar compositions of any of paragraphs Dthrough D.4.2 produced by the method of any of paragraphs A through B.4.

The disclosures of the references cited herein are incorporated byreference in their entireties.

Although the present invention has been shown and described withreference to the foregoing operational principles and illustratedexamples and embodiments, it will be apparent to those skilled in theart that various changes in form and detail may be made withoutdeparting from the spirit and scope of the invention. The presentinvention is intended to embrace all such alternatives, modificationsand variances that fall within the scope of the appended claims.

What is claimed is:
 1. A lignocellulosic sugar composition from asoftwood lignocellulosic biomass, wherein the lignocellulosic sugarcomposition, not including water, comprises at least 75% (wt/wt)monomeric lignocellulosic sugar, has a sulfur content less than about0.20% (wt/wt), has a metal ion content of at least 0.45% (wt/wt) andless than 3.0% (wt/wt), and has a ratio of monomeric glucose plusmonomeric mannose to at least one other monomeric lignocellulosic sugargreater than or equal to 93.6 and less than or equal to 280.7, whereinthe at least one other monomeric lignocellulosic sugar is selected fromthe group consisting of galactose and arabinose.
 2. The lignocellulosicsugar composition of claim 1, wherein the softwood lignocellulosicbiomass is Douglas fir.
 3. The lignocellulosic sugar composition ofclaim 2, wherein the lignocellulosic sugar composition, not includingwater, comprises at least 85% (wt/wt) monomeric lignocellulosic sugar.4. The lignocellulosic sugar composition of claim 2, wherein thelignocellulosic sugar composition, not including water, comprises atleast 90% (wt/wt) monomeric lignocellulosic sugar.
 5. Thelignocellulosic sugar composition of claim 2, wherein thelignocellulosic sugar composition is aqueous, and has a sugar titer ofat least 8.9% (wt/vol).
 6. The aqueous lignocellulosic sugar compositionof claim 5, wherein the aqueous lignocellulosic sugar composition has asulfur content of less than about 0.02% (wt/wt).
 7. The aqueouslignocellulosic sugar composition of claim 5, wherein the aqueouslignocellulosic sugar composition has a metal ion content of less thanabout 0.5% (wt/wt).
 8. The lignocellulosic sugar composition of claim 2,wherein the lignocellulosic sugar composition is free of furan and otherfermentation inhibitors.
 9. The lignocellulosic sugar composition ofclaim 2, wherein the lignocellulosic biomass from which thelignocellulosic sugar composition is produced is non-chemicallypretreated.
 10. The lignocellulosic sugar composition of claim 9,wherein the composition is produced by enzymatic hydrolysis of thenon-chemically pretreated lignocellulosic biomass.
 11. Thelignocellulosic sugar composition of claim 2, wherein the Douglas Fir isdebarked.
 12. A lignocellulosic sugar composition from a softwoodlignocellulosic biomass, wherein the lignocellulosic sugar composition,not including water, comprises at least 75% (wt/wt) monomericlignocellulosic sugar, has a sulfur content less than about 0.20%(wt/wt), has a metal ion content of at least 0.45% (wt/wt) and less than3.0% (wt/wt), and has a ratio of monomeric glucose to at least one othermonomeric lignocellulosic sugar greater than or equal to 77.9 and lessthan or equal to 233.7, wherein the at least one other monomericlignocellulosic sugar is selected from the group consisting of arabinoseand galactose.
 13. The lignocellulosic sugar composition of claim 12,wherein the softwood lignocellulosic biomass is Douglas fir.
 14. Thelignocellulosic sugar composition of claim 13, wherein the Douglas Firis debarked.
 15. The lignocellulosic sugar composition of claim 14,wherein the lignocellulosic sugar composition, not including water,comprises at least 85% (wt/wt) monomeric lignocellulosic sugar.
 16. Thelignocellulosic sugar composition of claim 14, wherein thelignocellulosic sugar composition is aqueous with a sugar titer of atleast 8.9% (wt/vol) and has a sulfur content of less than about 0.02%(wt/wt).
 17. The lignocellulosic sugar composition of claim 14, whereinthe lignocellulosic sugar composition is aqueous with a sugar titer ofat least 8.9% (wt/vol) and has a metal ion content of less than about0.5% (wt/wt).
 18. The lignocellulosic sugar composition of claim 14,wherein the lignocellulosic sugar composition is aqueous with a sugartiter of at least 8.9% (wt/vol) and is free of furan and otherfermentation inhibitors.